Brucella is a genus of Gram-negative bacteria which are the causative agents of brucellosis, an important disease of animals and humans. In animals, for example cattle, brucellosis causes abortions and in addition decreased meat and milk production. In humans, it produces intermittent debilitation with high fever which may resist antibiotic therapy and recur over a period of several years. The disease can therefore be the cause of serious health problems and substantial economic losses.
The species B. abortus, which is one example of this genus, remains a problem throughout the world. In South American countries, for example, up to 40% of cattle herds are affected by brucellosis, thousands of human cases are known, and economic losses are estimated at tens of millions of dollars annually. Other economically significant species include B. melitensis (which affects humans, sheep and goats), B. suis (which affects pigs, reindeer and humans), B. ovis (affecting sheep) and B. canis (affecting dogs and humans).
There have been advances in the development of vaccines against brucellosis. For example, in the case of B. abortus, there is common use of an attenuated live strain (S19) of B. abortus. However, two problems persist. The first is that, although the vaccine is effective, the protection it affords is not absolute, hence allowing some infection by field strains of B. abortus. The second problem is that, with current diagnostic techniques, vaccinated animals appear serologically similar to those infected. In Canada, limited vaccination of cattle by B. abortus S19 still occurs for export purposes. In the United States, 2.9 million cattle were vaccinated in 1985, in part because some of the southern states (Texas, Florida, Louisiana and Arkansas) still have high prevalences of brucellosis in their cattle populations. There is therefore an urgent need for a method of discriminating vaccinated from infected cattle.
To date, some discrimination is possible by setting limits for the amount of antibody against B. abortus produced using whole smooth-lipopolysaccharide (sLPS) complex in enzyme immunoassay (EIA or ELISA). FIG. 1 depicts graphically the distribution of cattle sera with antibodies having affinity or specificity for this antigen in one case study. In the Figure, the frequency or number of cattle, is plotted against the lower class limit (i.e. five sera with A.sub.414nm on the enzyme immunoassay of 1.00, 1.01, 1.02, 1.03 and 1.04 would be represented by a bar at 1.00 having a frequency of 5 sera). It can be seen that, in general, the vaccinates have readings below an A.sub.414nm of 0.50, while culture positives (i.e. B. abortus was isolated from these animals) have sera that gave an A.sub.414nm above 0.50 on this test. However, there is still considerable overlap, mostly in the 0-0.50 range but also some degree of overlap at higher readings (note the high positive readings of vaccinate sera around A.sub.414nm of 1.5).
The difficulty in differentiating vaccinates from infected cattle is again evident in a study that compared the sensitivities and specificities of five serodiagnostic tests used for the detection of bovine antibodies to B. abortus. The results of this study are given in the following Table 1.
TABLE 1 __________________________________________________________________________ COMPARATIVE ASSAY RESULTS Status Number of of Number of Positive Readings Cattle Cattle BPAT.sup.1 STAT.sup.2 CFT.sup.3 HIGT.sup.4 EIA.sup.5 __________________________________________________________________________ FREE LISTED.sup.a 1 non- 1067 12 (1.1%) 6 (0.6%) 0 (0%) 0 (0%) 1 (0.1%) vaccinated 2 vaccinated 76 6 (7.8%) 2 (2.6%) 0 (0%) 8 (10.5%) 3 (3.9%) REACTOR.sup.b 1 non- 798 74 (9.3%) 50 (6.3%) 9 (1.1%) 4 (0.5%) 5 (0.6%) vaccinated 2 vaccinated 253 20 (7.9%) 15 (5.9%) 5 (2.0%) 12 (4.7%) 14 (5.5%) INFECTED.sup.c 1 positive 174 152 (87.4%) 162 (93.1%) 159 (91.4%) 167 (96.0%) 162 (93.1%) __________________________________________________________________________ .sup.1 BPAT = buffered plate antigen test .sup.2 STAT = standard tube agglutination test .sup.3 CFT = complement fixation test .sup.4 HIGT = hemolysisin-gel test .sup.5 EIA = enzyme immunoassay .sup.a Free listed = cattle from herds certified in the previous year as being of brucellosis based on standard serological tests. .sup.b Reactor = cattle from herds containing two or more positive reactors based on standard serological tests, but herds in which no infected cattle have been indentified based on bacteriological culture. .sup.c Infected = cattle identified as infected by positive bacteriological culture
Table 1 shows that the buffered plate antigen test (BPAT, which is currently used as a screening test) recorded 1.1% of non-vaccinated and 7.8% of vaccinated cattle in Brucella-free herds as positive. The enzyme immunoassay (EIA, for further details see Nielsen and Wright, 1984, Agriculture Canada publication, ISBN 0-662-13421-4) greatly reduces the number of false-positive non-vaccinated cattle to 0.1% but the number for vaccinates remains high at 3.9%. The other serological tests show similar limitations. The complement fixation test (CFT) is an exception by having high specificity but its costs and technical manipulations make it unlikely to remain a routine method. Table 1 also shows that problem herds (those that persistently show reactors for no apparent cause) have a high number of false-positives for non-vaccinated and vaccinated cattle (i.e. 0.6% and 5.5% respectively for the EIA).
From the above data, it can be seen that present serodiagnostic tests are for the most part effective in discriminating Brucella-free from infected cattle. However, there is a small percentage of animals in the intermediate range (e.g. vaccinates with high titres or infected cattle with low titres of antibodies) that are impossible to classify reliably by standard serological methods. A small percentage of several million cattle is still a large population and since these animals may be highly valued, there is a great need for a test that will differentiate vaccinated cattle from B. abortus infected cattle.
T. J. G. Raybould and Shireen Chantler, J. Immunol. Methods, 30 (1979) pages 37-46 describe an immunofluorescent procedure in which antigenic extracts of B. abortus 544/W were coupled to Sepharose.RTM. beads. The crude sodium dodecyl sulfate (SDS) extract contained polysaccharide, lipopolysaccharide, proteins, etc., and when coupled to the Sepharose.RTM. beads showed discrimination between vaccinated and infected cattle, with, however, some overlap of results. In subsequent work (Raybould et al., Infect. Immun. 29 (1980) pages 435-441), they determined from inspection of the SDS extract that the active group for the discrimination was an antigen "X". As it bound to oxidised Sepharose.RTM., they concluded that antigen "X" contained amino groups and hence was a basic protein. The test given in that report involved hemagglutination, gave some overlap similar to that seen in other tests and in some instances was inconclusive.
It has also been reported (Diaz, R., P. Garatea, L. M. Jones, and I. Moriyon, 1979, Radial diffusion test with a Brucella polysaccharide antigen for differentiating infected from vaccinated cattle, J. Clin. Microbiol. 10:37-41; Jones, L. M., D. T. Berman, E. Moreno, B. L. Deyoe, M. J. Gilsdorf, J. D. Huber, and P. Nicoletti, 1980, Evaluation of a radial immunodiffusion test with polysaccharide B antigen for diagnosis of bovine brucellosis, J. Clin. Microbiol. 12:753-760), that there is a component of Brucella melitensis, termed "poly B" which can differentiate vaccinated from infected cattle, i.e. only the sera of the latter will precipitate "poly B" in agar gel immunodiffusion (or AGID). "Poly B" has been characterized by Moreno, E., S. L. Speth, L. M. Jones, and D. T. Berman, 1981, Immunochemical characterization of Brucella lipopolysaccharides and polysaccharides, Infect. Immun. 31:214-222, as a polysaccharide consisting primarily of glucose units. Our studies support these findings and we have now isolated "poly B" from B. melitensis 16M and found it to be essentially a polymer of glucose molecules. Some sera of infected cattle will react weakly with this component in AGID provided that 10% NaCl is added to the buffer (0.1 M sodium borate, pH 8.3). However, we have observed that while the principal component, "poly B" (the glucose polymer) is a poor antigen; the associated minor component, O-chain polysaccharide (containing 4,6-dideoxy-4-formamido-D-mannopyranose), is the immunodominant or "active ingredient" in these preparations. We have also found that reports of immunological identify between the O-chain polysaccharide and "poly B", the glucose polymer, (Moreno et al., 1981, Infect. Immun. 31; 214-222, Fernandez-Lago et al., 1982, Infect. Immun. 38, 778-780) were likely due to the latter containing O-chain as an impurity.